Chemoreceptors sensitive to the levels of CO2 or pH in the central nervous system are critical to the regulation of cardiorespiratory homeostasis. Disturbances in their function contribute to the morbidity and mortality associated with a number of diseases such as congenital central hypoventilation syndrome, chronic obstructive pulmonary disease, as well as sleep apnea and sudden infant death syndrome (SIDS). Despite the importance of central chemoreceptors in cardiorespiratory function, the relevance of specific chemoreceptor sites to respiratory regulation, and even the specific cell types serving this function (eg neurons or glia) remain controversial. Among the best supported sites for central chemoreception are the nucleus of the solitary tract (NTS) and the retrotrapezoid nucleus (RTN). To define the relative contributions of these sites to central chemoreception and the conditions under which they are active we will address two essential questions: 1) What are the molecular/biophysical bases of chemoreception for the candidate brainstem neurons? and 2) What are the pathways by which they provide input to central circuits controlling breathing? The proposed project will employ complementary in vivo and in vitro electrophysiological approaches, combined with neuroanatomical and molecular methods to define the molecular/biophysical basis of chemosensitivity within NTS and RTN cells, as well as their direct or indirect connections with brainstem respiratory circuits. In vitro recordings will take advantage of isolated neurons as well as slice recordings. Two-photon calcium imaging in acute slices will provide information on the response profiles of chemosensitive neurons. Neurons in these brainstem areas will be dissociated to allow careful electrophysiological characterization of the chemosensitive response to extracellular acidification. Effects of intracellular acidification will be determined using dual pipette patch clamp recordings with intracellular perfusion. Chemosensitive cells recorded in vivo will be juxtacellularly labeled to define their somatodendritic organization and local axonal arborization. Their brainstem targets will be determined by retrograde labeling. Homology of the filled neurons recorded in vivo with chemosensitive cells identified in vitro will be determined by comparing their content of the relevant pH sensitive ion channels, cell morphology including axonal projection, and related neurochemical markers. The impact of specific chemosensitive neuron types on the response to hypercapnia will be assessed during pharmacological blockade/stimulation of the target ion channels (identified in vitro) where specific antagonist/agonists exist (eg for TRPV1 channels implicated in our preliminary data). These experiments will shed light on one central question of animal physiology and may suggest new pharmacological targets for drug therapy. PUBLIC HEALTH RELEVANCE: Breathing is finely tuned by the partial pressures of arterial and brain carbon dioxide and failure of this tuning may lead to dramatic consequences such as the sudden Infant Death Syndrome (SIDS) or congenital central hypoventilation syndrome (CCHS); yet, the mechanisms underlying this regulation are not well understood. In particular, the circuitries and molecular mechanisms of CNS chemoreception remain the subjects of major debate. This proposal marshals electrophysiological, molecular and histochemical techniques to identify the neuronal types involved in central chemoreception and dissect the underlying molecular mechanisms.